The mouse mammary tumor virus (MMTV) is a B type retrovirus which causes primarily mammary carcinomas in mice. The MMTV long terminal repeat (LTR) has an unusual 1200 bp U3 region containing the transcription regulatory signals of the virus as well as a long open reading frame (orf) potentially encoding a protein of 36 kD. This orf is conserved in all known MMTV strains and the observed base substitutions give largely conservative or silent amino acid changes; this suggests conservation of protein function. In addition, a 1.7 kb spliced transcript which has the appropriate structure for orf mRNA has been identified in mammary glands and tumors from high mammary cancer mouse strains. Although orf has been reported to be a viral transactivator, this has not been confirmed, and the orf product has not been identified. Very recently we have overexpressed the unfused MMTV orf product from a recombinant baculovirus in insect cells. Because the 36 kD orf product appears to be a nuclear protein, we have speculated that orf is a positive or negative regulator of transcription which participates indirectly or cooperatively in MMTV-induced tumors. Several approaches will be used to test this hypothesis. In a genetic approach, we will construct an MMTV provirus which is incapable of orf synthesis for analysis of its replication potential in culture. Transgenic mice overexpressing the orf product will be assessed for the tissue-specific expression of orf, level of endogenous MMTV RNA, and the incidence of spontaneous tumors. In another approach, we will use the baculovirus- produced orf protein to develop polyclonal antibodies needed for identification and characterization of the orf product in mammalian cells expressing integrated MMTV proviruses. The baculovirus-produced protein also will be used for biochemical analysis. The intracellular location and posttranslational modifications of orf will be examined in insect cells, and partially purified orf will be used to evaluate the nucleic acid binding properties of the protein. Finally, the baculovirus orf protein will be employed in in vitro transcription reactions to determine if orf has either positive or negative effects on MMTV transcription. Because of the unusual nature of the MMTV orf and the general conservation of viral genetic information, it is likely that orf plays a unique role in MMTV replication or tumorigenesis.